Antibodies which are directed against the Marburg I polymorphism of factor VII-activating protease (FSAP), and their preparation and use

ABSTRACT

The invention relates to antibodies which are directed against the FSAP Marburg I variant and to their preparation and use, in particular in therapy and diagnosis. The antibodies are characterized by the fact that they bind specifically to the FSAP MR I variant but not to the FSAP wild-type protein or other mutant variants which are characterized by amino acid substitutions at other positions in the FSAP protein.

The invention relates to antibodies which are directed against theMarburg I polymorphism of the blood coagulation factor VII-activatingprotease (FSAP), and to their preparation and use.

The factor VII-activating protease (FSAP) is a plasma serine proteasewhich, in addition to its property of activating blood coagulationfactor VII also possesses plasminogen activator properties, that isprourokinase-activating properties [Roemisch et al. (1999) Haemostasis76: 292-299; EP 952 216 A2]. This suggests that FSAP plays a role both‘in the blood coagulation cascade and in the fibrinolytic system.

FSAP is present in human plasma at a concentration of approx. 12 μg/mland can be converted from the single-chain proenzyme into the activedouble-chain protease by means of autocatalysis. In addition to thewild-type sequence of the human FSAP gene, a variety of nucleotidepolymorphisms are known, with these polymorphisms in two cases alsoleading to a change in the amino acid sequence (EP 1 182 258 A1). Whatis termed the Marburg I polymorphism (also MR I mutation, allele orvariant) leads to a Gly/Glu amino acid substitution at position 534 ofthe proenzyme including the signal peptide (Gly/Glu 534) and results ina 50-80% reduction in the prourokinase-activating activity whereas theability to activate factor VII remains unaltered. Another polymorphism,i.e. what is termed the Marburg II polymorphism (also MR II mutation,allele or variant) leads to a Glu/Gln amino acid substitution atposition 370 of the proenzyme including the signal peptide (Glu/Gln370). However, the Marburg II mutation has no effect on theprourokinase-activating activity of the FSAP. An MR I polymorphism isfound in about 5% of the West-European population. Heterozygous carriersof the MR I polymorphism appear to be at a higher risk of developing acarotid stenosis than is the population on average [Willeit et al.(2003) Circulation 107: 667-670]. Consequently, FSAP, or the FSAP MR Ivariant, constitutes a potential marker for recognizing a dispositionfor atherosclerotic diseases.

In accordance with the prior art, it is possible to use a variety ofmethodological approaches for identifying individuals who are carryingat least one copy of the FSAP MR I variant (see EP 952 215 A1 and EP 1182 258 A1). One of the known methods is based on determining theprourokinase-activating potential of the FSAP in a sample. To do this, aspecific antibody, which is unable to distinguish between wild-type FSAPand the known FSAP variants, is coupled to a solid phase and incubatedwith the sample liquid. After prourokinase and chromogenic substratehave been added, the quantity of converted chromogenic substrate isdetermined as a measure of the prourokinase-activating activity of theFSAP. Carriers of the Marburg I polymorphism exhibit a prourokinaseactivity which is reduced by 50-80%. However, a reduction inprourokinase activity can also be due to the concentration of FSAP inthe sample being low. It is therefore particularly advantageous todetermine the FSAP antigen concentration in a sample in addition to theprourokinase activity. Monoclonal antibodies which enable FSAP to bedetected immunologically are known from the prior art. EP 1 182 258 A1describes two monoclonal antibodies which are derived from the hybridomacell lines DSM ACC2453 and, respectively, DSM ACC2454 and which wereobtained after immunizing mice with FSAP protein. Both the antibodiesbind the Marburg I and II variants as well as the FSAP wild-typeprotein. Other known FSAP antibodies bind equally to wild-type FSAP andto the known mutant variants, which means that the total content of FSAPantigen in a sample can be determined in a sandwich ELISA, for example(see also DE 100 23 923 A1). A reduction in prourokinase activity isonly a concrete indication of the presence of an FSAP MR I variant whenthis reduced activity is observed together with an FSAP antigenconcentration which is in the normal range.

However, none of the described methods provides reliable proof of thepresence of an FSAP MR I variant. However, in order to have recourse,for example, to appropriate prophylactic and therapeutic measures whenthe FSAP prourokinase activation potential is reduced, it is absolutelynecessary to diagnose the cause of the loss of function accurately.

It has thus far only been possible to detect the Gly/Glu amino acidsubstitution at position 534 of the proenzyme (Gly/Glu 534)unambiguously by sequencing the corresponding coding region in thegenomic DNA or the mRNA. A G/A base substitution in the genomicsequence, which can be detected at nucleotide position 1601 in the cDNA,is the genetic cause of the FSAP MR I polymorphism (see EP 1 182 258A1). Even if the DNA sequence analysis provides reliable results, theroutine laboratory has a need for established test methods which are aseconomic, reliable and rapid—as possible and which, in addition, can beimplemented automatically on available diagnostic equipment. Preferenceis given, in the main, to immunological detection methods or test assayssince they meet known criteria and are already used widely in laboratorydiagnostics.

The present invention was therefore based on the object of providing amethod and/or components which make it possible to specifically detectthe FSAP MR I variant using antibodies.

This object is achieved by the provision of the methods and objectsaccording to the invention which are described in the claims.

In particular, the object is achieved by the provision of antibodieswhich bind specifically to the FSAP MR I variant but not the FSAPwild-type protein or other mutant variants which are not characterizedby a Gly/Glu amino acid substitution at position 534 in the FSAPproenzyme. These antibodies form the basis for the direct immunologicaldetection and quantification of the FSAP MR I variant in plasma samplesfrom heterozygous carriers or homozygous individuals.

It has been found, surprisingly, that peptides which comprise at leastthe amino acid sequence Glu-Cys-Glu-Lys-Arg (SEQ ID NO:1), whichcorresponds to amino acids 532 to 536 in the FSAP MR I variant andconsequently comprises the Gly/Glu amino acid substitution at position534 of the proenzyme (Gly/Glu 534), are suitable for use as immunizingantigens for preparing FSAP MR I-specific antibodies. The peptidesaccording to the invention are distinguished by the fact that the FSAPMR I-specific Glu residue at position 534 is flanked, both N-terminallyand C-terminally, by at least two further amino acid residues.

Specific embodiments of the invention are explained in more detailbelow:

One part of the subject matter of this invention is represented bypeptides which comprise from 5 to 25 amino acids, such as from 5 to 20amino acids, or such as from 10 to 15 amino acids, and which arecharacterized in that they comprise the amino acid sequenceGlu-Cys-Glu-Lys-Arg (SEQ ID NO:1). In some embodiments of the invention,peptides comprise the amino acid sequenceTyr-Val-Tyr-Gly-Ile-Val-Ser-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr-Thr-Gln-Val-Thr-Lys-Phe(SEQ ID NO:2) or a fragment thereof which comprises at least the aminoacid sequence Glu-Cys-Glu-Lys-Arg (SEQ ID NO:1), such as a peptidecomprising the amino acid sequenceSer-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID NO:3).

Within the meaning of this invention, the term “peptides” encompassesacid amides which decompose into amino acids on hydrolysis, for exampleamino acid polymers such as polypeptides, oligopeptides, proteins orprotein fragments.

The peptides according to the invention can be used as immunizingantigen for preparing the antibodies according to the invention or elsefor purifying the antibodies according to the invention by means ofaffinity chromatography. Furthermore, the peptides according to theinvention can also be used in a method for quantitatively orqualitatively detecting an analyte, preferably the FSAP MR I variant.The peptides according to the invention can also be linked to, a solidphase and/or a component of a signal-generating system.

One aspect of the invention is represented by antibodies that bind tothe characterizing epitope of the FSAP MR I variant, i.e. to the aminoacid sequence Ser-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr (SEQID NO:3), which corresponds to amino acids 528-540 in the FSAP MR Ivariant.

Within the meaning of this invention, the term “antibody” is to beunderstood as signifying an immunoglobulin, for example animmunoglobulin of the IgA, IgD, IgE, IgG₁, IgG_(2a), IgG_(2b), IgG₃,IgG₄ or IgM class or subclass. An antibody possesses at least onebinding site (frequently termed paratope) for an epitope (frequentlyalso termed antigenic determinant) on an antigen or hapten. Such anepitope is characterized, for example, by its spatial structure and/orthe presence of polar and/or apolar groups. The binding site of theantibody is complementary to the epitope. The antigen-antibody reactionor the hapten-antibody reaction functions in accordance with what istermed the “key-keyhole principle” and is as a rule highly specific,i.e. the antibodies are able to distinguish small differences in theprimary structure, in the charge, in the spatial configuration and inthe steric arrangement of the antigen or hapten. In particular, what aretermed the “complementarity-determining regions” of the antibodycontribute to binding the antibody to the antigen or hapten.

The term “antigens” encompasses monovalent and polyvalent antigens. Apolyvalent antigen is a molecule or a molecular complex to which morethan one immunoglobulin is able to bind simultaneously whereas only onesingle antibody can bind at any one time to a monovalent antigen. Ahapten is usually the name given to a molecule which is not immunogenicon its own but which, for immunization purposes, is usually bound to acarrier.

Within the meaning of this invention, the term antibodies is to beunderstood as signifying not only complete antibodies but also,expressly, antibody fragments such as Fab, Fv, F(ab′)₂ and Fab′; as wellas chimeric, humanized, bispecific or oligospecific, or single-chainantibodies; and, in addition, also aggregates, polymers and conjugatesof immunoglobulins and/or their fragments provided their properties withregard to binding to the antigen or hapten are retained. Antibodyfragments can be prepared, for example, by cleaving antibodiesenzymatically with enzymes such as pepsin or papain. Antibodyaggregates, antibody polymers and antibody conjugates can be generatedby many different methods, for example by heat treatment, by reactionwith substances such as glutaraldehyde, by reaction withimmunoglobulin-binding molecules, by biotinylation of antibodies andsubsequent reaction with streptavidin or avidin, etc.

Within the meaning of this invention, an antibody can be a monoclonalantibody or a polyclonal antibody. The antibody can have been preparedin accordance with the customary methods, for example by immunizing thehuman subject or an animal, such as a mouse, a rat, guinea pig, rabbit,horse, donkey, sheep, goat or hen also Messerschmid (1996) BIOforum 11:500-502], ‘and then isolating the antiserum; or by, establishinghybridoma cells and subsequently purifying the secreted antibodies; orby cloning and expressing the nucleotide sequences, or modified versionsthereof, which encode the amino acid sequences which are responsible forthe binding of the natural antibody to the antigen and/or hapten.

Antibodies according to the invention include, for example, antibodieswhich bind to a peptide of from 5 to 25 amino acids, such as from 5 to20 amino acids, or such as from 10 to 15 amino acids, and whichcomprises the FSAP MR I-specific amino acid sequence Glu-Cys-Glu-Lys-Arg(SEQ ID NO:1). Some antibodies of this invention bind specifically topeptides comprising the amino acid sequenceSer-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID NO:3) or toa fragment of this peptide which comprises at least the amino acidsequence Glu-Cys-Glu-Lys-Arg (SEQ ID NO:1).

The antibodies which are produced by the hybridoma cell lines

a) Mab (mouse) directed against ECE-KLH 2004-9/014 (1),

b) Mab (mouse) directed against ECE-KLH 2004-9/026 (2),

c) Mab (mouse) directed against ECE-KLH 2004-35/05 (1),

d) Mab (mouse) directed against ECE-KLH 2004-34/08 (2) or

e) Mab (mouse) directed against ECE-KLH 2004-151/013 (2)

are also embodiments of this invention. The hybridoma cell lines a) toc) were deposited on Aug. 11, 2004 in the DSMZ—Deutsche Sammlung vonMikroorganismen und Zellkulturen [German collection of microorganismsand cell cultures] GmbH, MascheroderWeg 1b, 38124 Braunschweig, Germany,under the receipt numbers a) DSM ACC2675 b) DSM ACC2676 and c) DSMACC2674. The hybridoma cell lines d) and e) were deposited on May 19,2005 in the abovementioned depository institution under the receiptnumbers d) DSM ACC2725 and e) DSM ACC2726.

Another part of the subject matter of this invention relates to specificbinding partners which bind to an epitope which is recognized by anantibody according to the invention.

A “specific binding partner” is to be understood as being a member of aspecific binding pair. The members of a specific binding pair are twomolecules which in each case possess at least one structure which iscomplementary to a structure of the other molecule, with the twomolecules being able to bind to each other by means of the complementarystructures binding. The term molecule also encompasses molecularcomplexes such as enzymes which consist of apoenzyme and coenzyme,proteins which consist of several subunits, lipoproteins which consistof protein and lipids, etc. Specific binding partners can be naturallyoccurring substances or else substances which are, for example, preparedby means of chemical synthesis, microbiological techniques and/orrecombinant methods. The following may, for example, be mentioned inorder to illustrate the term specific binding partner but without thisbeing understood as a limitation: thyroxine-binding globulin,steroid-binding proteins, antibodies, antigens, haptens, enzymes,lectins, nucleic acids, repressors, oligonucleotides andpolynucleotides, protein A, protein G, avidin, streptavidin, biotin,complement component Clq, nucleic acid-binding proteins, etc. Examplesof specific binding pairs are: antibody-antigen, antibody-hapten,operator-repressor, nuclease-nucleotide, biotin-avidin,lectin-polysaccharide, steroid-steroid-binding protein, activecompound-active compound receptor, hormone-hormone receptor,enzyme-substrate, IgG-protein A, complementary oligonucleotides orpolynucleotides, etc.

As a result of providing the antibodies according to the invention, itis now possible for the skilled person, for example by means ofcompetition experiments (see also Peters et al. (1985) MonoklonaleAntikörper [Monclonal Antibodies], Springer Verlag, Chapter 12.2“Epitope analysis”], to identify other specific binding partners, withantibodies being expressly included, which bind to the epitope of anantibody according to the invention. Thus, specific binding partners canby now be selected using phage display libraries, by way of syntheticpeptide databases, or using recombinatorial antibody libraries [Larrick& Fry (1991) Human Antibodies and Hybridomas 2: 172-189].

This invention also relates to an antibody according to the inventionwhich is linked to a solid phase and/or a component of asignal-generating system.

Within the meaning of this invention, the term “solid phase” means anobject which consists of a porous and/or nonporous, as a rulewater-insoluble, material and can have a very wide variety of forms, forexample those of vessels, tubes, microtitration plates, spheres,microparticles, rods, strips, filter paper, chromatography paper, etc.As a rule, the surface of the solid phase is hydrophilic or can be madehydrophilic. The solid phase can consist of a very wide variety ofmaterials, for example of inorganic and/or organic materials, ofsynthetic materials, of naturally occurring materials and/or of modifiednaturally occurring materials. Examples of solid phase materials arepolymers, such as cellulose, nitrocellulose, cellulose acetate,polyvinyl chloride, polyacrylamide, crosslinked dextran molecules,agarose, polystyrene, polyethylene, polypropylene, polymethacrylate ornylon; ceramic, glass or metals, in particular precious metals such asgold and silver; magnetite; mixtures or combinations thereof; etc.Cells, liposomes and phospholipid vesicles are also covered by the termsolid phase.

The solid phase can also possess a coating consisting of one or morelayers, for example of proteins, carbohydrates, lipophilic substances,biopolymers or organic polymers, or mixtures thereof, in order, forexample, to suppress or prevent the nonspecific binding of sampleconstituents to the solid phase or in order, for example, to achieveimprovements with regard to the suspension stability of particular solidphases, with regard to storage stability, with regard to dimensionalstability or with regard to resistance to UV light, microbes or otheragents having a destructive effect.

A “signal-generating system” can be one or more components with at leastone of the components being a detectable label. A label is to beunderstood as being any molecule which itself produces a signal or whichis able to induce the production of a signal, for example a fluorescentsubstance, a radioactive substance, an enzyme or a chemiluminescentsubstance. The signal can, for example, be detected or measured usingthe enzyme activity, the luminescence, the light absorption, the lightscattering, the emitted electromagnetic or radioactive radiation or achemical reaction.

A label may itself be able to generate a detectable signal, such that nofurther components are required. Many organic molecules absorbultraviolet and visible light, resulting in these molecules being ableto reach an excited energy state and to emit the absorbed energy in theform of light which is of a different wavelength from that of theincident light. Yet other labels can directly generate a detectablesignal, for example radioactive isotopes or dyes.

Yet other labels require additional components for generating thesignal, i.e., in such a case, the signal-producing system includes allthe components, such as substrates, coenzymes, quenchers, accelerators,additional enzymes, substances which react with enzyme products,catalysts, activators, cofactors, inhibitors, ions, etc., which arerequired for producing the signal.

Examples of suitable labels [see also EP 0 515 194 A2; U.S. Pat. No.5,340,716; U.S. Pat. No. 5,545,834; Bailey et al. (1987) J.Pharmaceutical & Biomedical Analysis 5: 649-658] are enzymes, includinghorseradish peroxidase, alkali phosphatase, glucose 6-phosphatedehydrogenase, alcohol dehydrogenase, glucose oxidase, β-galactosidase,luciferase, urease and acetylcholine esterase; dyes; fluorescentsubstances, including fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanine, ethidium bromide,5-dimethylamino-naphthalene-1-sulfonyl chloride and fluorescent chelatesof rare earths; chemiluminescent substances including luminol,isoluminol, acridinium compounds, olefin, enolether, enamine,arylvinylether, dioxene, arylimidazole, lucigenin, luciferin andaequorin; sensitizers, including eosin, 9,10-dibromoanthracene,methylene blue, porphyrin, phthalocyanine, chlorophyll and Rose Bengal;coenzymes; enzyme substrates; radioactive isotopes, including ¹²⁵I, ¹³¹I¹⁴C, ³H, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁹Fe, ⁵⁷Co and ⁷⁵Se; particles, includingmagnetic particles or particles, preferably latex particles, whichthemselves can be labeled with, for example, dyes, sensitizers,fluorescent substances, chemiluminescent substances, isotopes or otherdetectable labels; sol particles including gold sols or silver sols;liposomes or cells which can themselves be labeled with detectablelabels; etc.

A signal-generating system can also comprise components which, when inspatial proximity to each other, are able to enter into a detectableinteraction, for example components in the form of energy donors andenergy recipients, such as photosensitizers and chemiluminescentsubstances (EP 0 515 194 A2), photosensitizers and fluorophores (WO95/06877), radioactive iodide¹²⁵ and fluorophores [Udenfriend et al.(1985) Proc. Natl. Acad. Sci. 82: 8672-8676], fluorophores andfluorophores [Mathis (1993) Clin. Chem. 39: 1953-1959] or fluorophoresand fluorescence quenchers (U.S. Pat. No. 3,996,345). An interactionbetween components also includes the direct transfer of energy betweenthe components, for example by means of light radiation or electronradiation and also by way of short-lived reactive chemical molecules.Interactions also include processes in which the activity of a componentis inhibited or augmented by one or more other components, for examplethe inhibition of, or increase in, the enzyme activity or the inhibitionof, increase in or change in (e.g. wavelength shift, polarization) theelectromagnetic radiation which is emitted by the affected component.The interaction between the components also includes enzyme cascades. Inthis case, the components are enzymes at least one of which provides thesubstrate for another, thereby resulting in the coupled substratereaction taking place at a maximum or minimum reaction rate. As a rule,an effective interaction between the components takes place when thecomponents are spatially adjacent to each other, that is, for example,within a distance range of a few μm, in particular within a distancerange of less than 600 nm, preferably less than 400 nm, veryparticularly preferably less than 200 nm.

Microparticles are frequently used as the solid phase and/or the label.Within the meaning of this invention, the term “microparticles” is to beunderstood as signifying particles which have an approximate diameter ofless than 20 nm and not more than 20 μm, customarily between 40 nm and10 μm, such as between 0.1 and 10 μm, between 0.1 and 5 μm, or between0.15 and 2 μm. The microparticles can have a regular or irregular shape.They can be spheres, spheroids or spheres having cavities or pores ofgreater or lesser size. The microparticles can also comprise organicmaterial or inorganic material or a mixture or combination of the two.They can comprise a porous or nonporous, a swellable or nonswellablematerial. While, in principle, the microparticles can be of any density,some particles used in the invention have a density, such as from about0.7 to about 1.5 g/ml, which approaches the density of water. Themicroparticles can be suspended in aqueous solutions, with theirsuspensions being stable for as long as possible. It is possible forthem to be transparent, partially transparent or nontransparent. Themicroparticles can comprise several layers such as, for example, whatare termed core-and-shell particles having a core and one or moreenveloping layers.

The term microparticles encompasses, for example, dye crystals, metalsols, silica particles, glass particles, magnetic particles, polymericparticles, oil drops, lipid particles, dextran and protein aggregates.In some embodiments, microparticles can be suspended in aqueoussolutions and comprise water-insoluble polymer material, such assubstituted polyethylenes. In some embodiments, microparticles compriselatex particles of, for example, polystyrene, acrylic acid polymers,methacrylic acid polymers, acrylonitrile polymers,acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate,polyvinylpyridine or vinyl chloride-acrylate. In some embodiments, latexparticles possess reactive groups, such as carboxyl, amino or aldehydegroups, at their surface, with these groups enabling specific bindingpartners, for example, to be bonded covalently to the latex particles.The preparation of latex particles is described, for example, in EP 0080 614, EP 0 227 054 and EP 0 246 446.

The term “linked” is to be understood in a broad manner and encompasses,for example, a covalent bond and a noncovalent bond, a direct linkageand an indirect linkage, adsorption to a surface and inclusion in aninvagination or a cavity, etc. In the case of a covalent bond, theantibodies or binding partners are bound to the solid phase or to thelabel by way of a chemical bond. Examples of a noncovalent bond aresurface adsorption, inclusion in cavities or the binding of two specificbinding partners. In addition to a direct linkage to the solid phase orthe label, the antibodies or binding partners can also be boundindirectly to the solid phase or the label by way of specificinteraction with other specific binding partners (see also EP 0 411 945A2). This will be illustrated in more detail with the aid of examples:the biotinylated antibody can be bound to the label by way oflabel-bound avidin, or a fluorescein-antibody conjugate can be bound tothe solid phase by way of solid phase-bound anti-fluorescein antibodies,or the antibody can be bound to the solid phase or the label by way ofimmunoglobulin-binding proteins.

This invention furthermore relates to specific binding partners orantibodies according to the invention which are used as an in-vitrodiagnostic agent or as a constituent of an in-vitro diagnostic agent. Inthe case of an in-vitro diagnostic agent, the analyte to be detected,e.g. the FSAP Marburg I variant, is detected, or its concentration orquantity is determined, in a sample outside a living human or animalbody.

Within the meaning of the invention, a “sample” is to be understood assignifying the material which is suspected of containing the substanceto be detected (for examples of the term “analyte”, see EP 0 515 194 A2,pages 8-15). The term sample encompasses, for example, biological fluidsor tissues from humans and animals, in particular, such as blood,plasma, serum, sputum, exudate, bronchoalveolar lavage, lymph fluid,synovial fluid, seminal fluid, vaginal mucus, feces, urine, cerebralspinal fluid, hair, skin and tissue samples or sections. It alsoencompasses cell culture samples, plant liquids or tissues, forensicsamples, water and effluent samples, nutrients and pharmaceuticals.Where appropriate, the samples have to be pretreated in order to makethe analyte accessible to the detection method or in order to removeinterfering sample constituents. Such a pretreatment of samples mayinvolve separating off and/or lysing cells, precipitation, thehydrolysis or denaturation of sample constituents such as proteins, thecentrifugation of samples, the treatment of the sample with organicsolvents such as alcohols, in particular methanol; and the treatment ofthe sample with detergents. The sample is frequently transferred intoanother, usually aqueous, medium, which will, if at all possible, notinterfere with the detection method.

The antibodies according to the invention can be used in a method forquantitatively or qualitatively detecting an analyte, preferably theFSAP Marburg I variant, in a sample.

In the case of a quantitative detection, the quantity, the concentrationor the activity (e.g. enzyme activity) of the analyte in the sample ismeasured. The term “quantitative detection” also includessemi-quantitative methods which are only able to detect the approximatequantity, concentration or activity of the analyte in the sample or areonly able to be used to give a relative indication of the quantity,concentration or activity. A qualitative detection is to be understoodas detecting whether the analyte is at all present in the sample orindicating that the concentration or activity of the analyte in thesample is below or above one particular threshold value or severalparticular threshold values.

The invention consequently also relates to methods for quantitatively orqualitatively detecting an analyte, preferably FSAP Marburg I variant,in a sample, and to reagents which are suitable for this purpose.

Binding tests in which the specific binding of analyte to be detected toanalyte-specific binding partners can be used to draw conclusions withregard to the presence, absence or quantity of the analyte in a sampleare frequently employed for detecting analytes. Immunoassays or methodsin which oligonucleotides or polynucleotides are hybridized are examplesof binding tests.

What are termed “heterogeneous binding tests” are characterized by oneor more separation steps and/or washing steps. The separation can beeffected, for example, by means of immunoprecipitation, precipitationwith substances such as polyethylene glycol or ammonium sulfate,filtration, magnetic separation, or binding to a solid phase. Such a“solid phase” consists of porous and/or nonporous, as a rulewater-insoluble, material. It can have a very wide variety of differentforms, such as that of vessels, tubes, microtitration plates, spheres,microparticles, rods, strips, filter paper or chromatography paper, etc.In the case of heterogeneous binding tests in sandwich format, one ofthe analyte-specific binding partners is as a rule bound to a solidphase and serves for separating off the “analyte/analyte-specificbinding partner” binding complex from the liquid phase while the otheranalyte-specific binding partner carries a detectable label, e.g. anenzyme, or a fluorescence or chemiluminescene label, etc., for detectingthe binding complex. These test methods are further subdivided into whatare termed one-step sandwich tests, in which the two specific bindingpartners are incubated simultaneously with the sample, and into two-stepsandwich tests, in which the sample is first of all incubated with thesolid-phase reagent and, after a separation and washing step, the solidphase-bound binding complex composed of analyte and analyte-specificbinding partner is incubated with the detection reagent.

In the case of “homogeneous binding tests”, the components of thesignal-generating system which are bound to the“analyte/analyte-specific binding partner” complex and those which arefree are not separated. The test mixture, which contains theanalyte-specific binding partners, the signal-generating components andthe sample, is measured, and the corresponding measurement signaldetermined, after, or even during, the binding reaction without anyfurther separation and/or washing step. Many turbidimetric ornephelometric methods, in which the analyte-specific binding partnersemployed for the detection can be linked to latex particles, such asEMIT® tests; CEDIA® tests; fluorescence-polarization immunoassays;luminescent oxygen channeling immunoassays [LOCI®, see EP 0 515 194 A23;Ullman et al. (1994) Proc. Natl. Acad. Sci. 91: 5426-5430; Ullman et al.(1996) Clinical Chemistry 42: 1518-1526] etc., are examples ofhomogeneous immunoassays [see also Boguslaski & Li (1982) AppliedBiochemistry and Biotechnology 7: 401-414]. In a homogeneous sandwichimmunoassay, such as a nephelometric latex test, the antibody reagentsare incubated with the sample and the signal is measured during and/orafter the incubation without a separation or washing step being carriedout prior to the measurement. Expressed in other words: theantibody-bound analyte is not separated from the free analyte or fromantibodies which have not bound any analyte.

The antibodies according to the invention are particularly suitable foruse in homogeneous binding tests.

Homogeneous and heterogeneous binding tests can also be carried out inthe form of what is termed a “sandwich assay”. In this case, the analyteis, for example in the case of a heterogeneous binding test, bound by asolid phase-linked, analyte-specific binding partner and by ananalyte-specific binding partner which is linked to a component of asignal-generating system. In sandwich immunoassays, antibodies orantigens or haptens can be the analyte-specific binding partners.

The “indirect immunoassay” is another special embodiment of aheterogeneous or homogeneous binding test. In this case, the analyte isan antibody. One of the analyte-specific binding partners is the antigenor, for example, the peptides according to the invention, or a modifiedantigen of the antibody to be detected (=analyte), and the otheranalyte-specific binding partner is as a rule an immunoglobulin-bindingprotein such as an antibody which is able to specifically bind theantibody to be detected (=analyte).

In a homogeneous or heterogeneous “competitive binding test”, sampleanalyte and reagent analyte compete for binding to a limited number ofanalyte-specific binding partners. The reagent analyte is, for example,a “modified analyte” such as a labeled analyte, an analyte fragment,such as the peptides according to the invention, or an analyte analog.The following examples illustrate the principle: (i) sample analytecompetes with reagent analyte, which is linked with a component of asignal-generating system, for binding to solid phase-linked,analyte-specific binding partners, or (ii) sample analyte competes withsolid phase-linked analyte (=reagent analyte) for binding toanalyte-specific binding partners which are linked to a component of asignal-generating system.

The antibodies according to the invention can also be used to detect theFSAP Marburg I variant employing methods such as Western blotting, dotblotting, immunoelectrophoresis, immunofixation electrophoresis,electroimmunodiffusion, immunoprecipitation, radial immunodiffusion,immunofixation, immunochromatography, latex agglutination, turbidimetricor nephelometric test, homogeneous or heterogeneous binding test,one-step or two-step test, sandwich test, indirect test, competitivetest, point-of-care tests, etc. These, and other, detection methods aredescribed, for example, in “Labor und Diagnose [Laboratory andDiagnosis]”, ed. L. Thomas, TH-Books Verlagsgesellschaft mbH, Frankfurt,1998, chapter 60, or in “Laboratory Techniques in Biochemistry andMolecular Biology—An Introduction to Radioimmunoassay and RelatedTechniques”, ed. T. Chard, Elsevier, Amsterdam, 1987.

The term “point-of-care tests” or “POC tests” encompasses tests in whichno separate analytical or measuring equipment is required for carryingout the test or evaluating the test. In many cases, POC tests are basedon immunochromatographic methods, immunocomplex separation by means offiltration and/or immunofixation techniques. POC tests are intended, inparticular, for measurements carried out on the spot, for example at thepatient's bedside or at home, and for the emergency doctor and/or theregistered physician and less for the large laboratory. POC tests can,in particular, also be carried out by individuals who do not have anydetailed medical training and experience in the field of laboratorymedicine. Within the meaning of this invention, the term “POC tests” isalso to be understood as including what are termed home tests or OTCtests which may be carried out by medical lay persons, such as, forexample, the various pregnancy tests which are marketed for use at home.Other POC tests relate, for example, to detecting cardiac infarctionmarkers, drugs, pharmaceuticals, infection markers and inflammationmarkers. In many POC tests, specific binding partners are linked, orbecome linked during the course of implementing the test, to or onfilter or chromatographic strips or discs. A positive or negativedetection reaction can, for example, be linked to the appearance ornonappearance of a color band in a particular test field and/or theappearance or nonappearance of a particular symbol, for example a “+” ora “−” and/or the intensity of the respective measurement signal.

A POC test for the FSAP Marburg I variant can, for example, be set up asfollows: the sample and labeled antibodies which are able to bind to theFSAP Marburg I variant but not to wild-type FSAP or other FSAP variantsare loaded onto a test strip. Examples of suitable labels are coloredlatex particles, colloidal gold, enzymes, etc. If the FSAP Marburg Ivariant is present in the sample, FSAP MR I/antibody complexes will beformed. These complexes move, for example by means of capillary forces,in the direction towards a region in which antibodies which are able tobind to other FSAP MR I epitopes, and which are fixed, or become fixedduring the course of the test procedure (e.g. by way of a biotin-avidinbridge), for example in the form of a band, are located. The labeledFSAP MR I/antibody complexes are bound in this region and form asandwich complex together with the fixed antibodies. In this case, theintensity of the label signal is proportional to the concentration ofFSAP MR I in the sample. In the case of a competitive POC test method,antibody fragments can, for example, be fixed, or become fixed duringthe course of the test procedure, in a region of the test strip. Thisfixed antibody would compete with the FSAP Marburg, I variant from thesample for binding to labeled anti-FSAP MR I antibodies. Alternatively,fixed anti-FSAP Marburg I variant antibodies and labeled FSAP MR Iprotein, or the peptides according to the invention, can also beemployed for setting up a competitive FSAP Marburg I variant test.

One embodiment of the method according to the invention is anephelometric or turbidimetric test, for instance, a test which employsantibodies according to the invention, optionally linked to a solidphase such as microparticles (including latex particles).

Another part of the subject matter of the invention is a test kit whichcomprises one or more of the antibodies and/or peptides according to theinvention. Such a kit customarily comprises all or only some componentsof a test in packaged form. The antibodies and/or peptides according tothe invention can, for example, be linked to one or more solid phasesand/or one or more components of a signal-generating system. The testkit can, for example, comprise standards, controls, calibrators andother reagents, such as buffers, washing solutions, measurementsignal-initiating solutions and/or enzyme substrate, cuvettes, pipettesand/or test instructions.

Reconstitutable lyophilized preparations, such as liquid preparationswhich either contain a defined quantity of FSAP Marburg I protein in anative or recombinant form or a defined quantity of a peptide accordingto the invention which comprises the amino acid sequenceGlu-Cys-Glu-Lys-Arg (SEQ ID NO:1) are suitable for use as standards,controls or calibrators in methods for quantitatively or qualitativelydetecting the FSAP Marburg I variant using specific binding partners.Molecules comprising a peptide moiety of from 5 to 25 amino acidscomprising the amino acid sequence Glu-Cys-Glu-Lys-Arg (SEQ ID NO:1) aswell as another moiety of from 5 to 15 amino acids and which comprisesthe amino acid sequence Glu-Glu-Phe-His-Glu (SEQ ID NO:4) are likewisesuitable. The latter peptide moiety may comprise the amino acid sequenceGln-Asp-Leu-Lys-Lys-Glu-Glu-Phe-His-Glu-Gln-Ser-Phe-Arg-Val (SEQ IDNO:5) or of a fragment thereof comprising the sequenceGlu-Glu-Phe-His-Glu (SEQ ID NO:4). The amino acid sequenceGlu-Glu-Phe-His-Glu (SEQ ID NO:4) corresponds to the amino acidpositions 383 to 387 in the FSAP proenzyme and represents an epitopewhich is present both in the wild-type FSAP protein and in the FSAPMarburg I variant. Since the fusion peptides according to the inventionare bound both by binding partners having specificity for the FSAPMarburg I polymorphism, for example, by the antibodies according to theinvention, and by binding partners having specificity for theGlu-Glu-Phe-His-Glu-containing epitope of the FSAP proenzyme, forexample by monoclonal antibodies which are formed by the hybridoma cellline which was deposited in the DSMZ—Deutsche Sammlung vonMikroorganismen and Zellkulturen [German collection of microorganismsand cell cultures] GmbH in Braunschweig, Germany, under the receiptnumber DSM ACC2453 (see EP 1 182 258 A1), they are particularly suitablefor standardizing, calibrating or controlling quantitative orqualitative sandwich test methods. The fusion peptides can be preparedby means of an appropriate peptide synthesis. It is likewise possible tolink the FSAP Marburg I epitope and the wild-type epitope together usinghomobifunctional crosslinkers such as glutaraldehyde or bi-NHS esters orusing heterobi-functional crosslinkers such as N-hydroxysuccinimide(NHS)-X-maleimide or NHS-X-haloacetyl,N-γ-maleimidobutyryloxysuccinimide ester (GMBS), N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB) and heterobifunctional reagentswhich generate a free SH group, such as iminothiolane, N-succinimidylS-acetylthioacetate (SATA), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 4-succinimidyloxycarbonyl-methyl-{acute over(α)}-(2-pyridyldithio) toluene (SMPT), or to conjugate the two epitopeswith a shared carrier, such as ovalbumin, albumin, keyhold limpethemocyanin (KLH) or bovine alpha-lactalbumin, as well as polymers suchas dextran, polyethylene glycol, polyacrylamide orpoly-d-glutamine-d-lysine, etc. The peptide can, for example, be boundto these carriers using carbodiimide or glutaraldehyde or else using aheterobifunctional reagent, such as N-maleimidobutyryl-oxysuccinimideester (GBMS), which can also act as a spacer. For further examples andfor coupling methods, see also Wong, S. (1993) Chemistry of ProteinConjugation and Cross-Linking, CRC Press Inc., Boca Raton.

One test kit according to the invention comprises antibodies accordingto the invention and/or peptides according to the invention which arelinked to latex particles.

Another test kit according to the invention comprises a plasma productwhich consists of a pool of at least five, preferably of more than 20,human plasmas from heterozygous FSAP Marburg I donors. Heterozygousdonors can be identified using known screening methods (see EP 1 182 258A1) and, naturally, also using the antibodies according to theinvention. Such an FSAP Marburg I plasma pool can be used, for example,as a standard, with a reference value, such as 100% or 1 PEU (plasmaequivalent units)/ml, being defined for the pool. Such a pool, ordifferent dilutions of the plasma pool, can be used for setting upsubstandards (e.g. peptide standards) or for defining a cut-off valueand a gray region, which may possibly be required, for detecting theFSAP Marburg I variant antigen.

The antibodies and the peptides according to the invention can also beused for affinity chromatography. The term “affinity chromatography” isto be understood as meaning a method for purifying and isolatingsubstances, in particular biopolymers, which is based on the fact thatmany substances are able to enter into a selective, noncovalent,reversible binding with binding partners which are specific for them.The principle of the method is that the specific binding partner isbonded, as a rule covalently, to an insoluble matrix (e.g. porousglasses, or gels based on agarose, cellulose, dextran, polymer orsilica) and brought into contact with a sample which contains thesubstance. Due to its specific interaction with the matrix-bondedspecific binding partner, the sought-after substance is immobilized andretained while all the other substances present in the sample areseparated off by elution. The sought-after substance is then releasedfrom the matrix using a suitable eluent which abolishes the noncovalentbond between the substance and the specific binding partner (see also E.Buddecke, 1989, Grundrisse der Biochemie [Outlines of biochemistry],Walter de Gruyter, chapter 7 “Proteine [Proteins]”).

Another part of the subject matter of this invention encompassesantibodies according to the invention or peptides according to theinvention in a pharmaceutically tolerated, sterile injection medium. Apharmaceutically tolerated, sterile injection medium is to beunderstood, for example, as being an organism-free, pyrogen-freesolution, for example saline or another electrolyte solution as iscustomarily used for the intravenous, intramuscular, intraperitoneal orsubcutaneous administration of pharmaceuticals, vaccines or contrastagents.

Yet another part of the subject matter of this invention is the use ofthe antibodies according to the invention as a diagnostic agent or as aconstituent of a diagnostic agent.

Another part of the subject matter of this invention is a method forpreparing an antibody according to the invention, which method ischaracterized in that one or more peptides of from 5 to 25 amino acids,such as from 5 to 20 amino acids, or such as from 10 to 15 amino acids,and which comprise the amino acid sequence Glu-Cys-Glu-Lys-Arg (SEQ IDNO: 1) are used for the immunization. The immunizing antigens whichcomprise this method according to the invention are peptides which havethe amino acid sequenceSer-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID No: 3) orfragments which at least comprise the amino acid sequenceGlu-Cys-Glu-Lys-Arg (SEQ ID NO: 1).

The antibodies according to the invention can also be prepared by usingnaturally occurring and/or recombinant FSAP MR I protein, or fragmentsthereof, which contain the FSAP MR I-specific polymorphism.

The peptides which are used as immunizing antigens can be used for theimmunization in unbound form and/or in carrier-bound form.

Examples of typical carriers are proteins, such as ovalbumin, albumin orkeyhole limpet hemocyanin (KLH), or polymers, such as polyethyleneglycol, polyacrylamide or poly-d-glutamine-d-lysine. The peptides can,for example, be bonded to these carriers using carbodiimide orglutaraldehyde or else using a heterobifunctional reagent, such asN-maleimidobutyryl-oxysuccinimide ester (GBMS), which can also act as aspacer. For further examples and coupling methods, see also Wong, S.(1993) Chemistry of Protein Conjugation and Cross-Linking, CRC Press,Inc., Boca Raton.

A preferred method for preparing the peptides according to the inventionwhich are used, inter alia, as immunizing antigens is that ofsolid-phase synthesis, with a multiplicity of copies of a peptide beingsynthesized on a lysine nucleus [see also Tam J. P. (1988) Proc. Natl.Acad. Sci. USA 85: 5409-5413]. The peptide synthesis is preferablycarried out in accordance with a standard protocol and using automatedequipment as is offered for sale, for example, by Applied Biosystems(USA). These multimeric peptides can subsequently be bonded to a carrierprotein.

The immunizing antigen can, for example, be taken up inphosphate-buffered saline and treated with Immune Easy Mouse Adjuvant.This emulsion can then be administered, for example intradermally,intraperitoneally and/or subcutaneously, to an animal, for example arabbit, a mouse, a rat, a guinea pig, a horse, a donkey, a sheep, agoat, a chicken, etc. Booster injections, with it also being possible toemulsify the immunizing antigen with incomplete Freund's adjuvant, mayhelp to increase the immune response.

Polyclonal antibodies according to the invention can be isolated fromthe antiserum of the immunized animals and can be further purified bymeans of an affinity chromatography through a matrix to which, forexample, the FSAP Marburg I variant or the peptides employed asimmunizing antigen has/have been bonded.

In order to produce monoclonal antibodies according to the invention,the immune cells of immunized animals, such as a mouse or a rabbit, are,in accordance with the well-known methods [see also Harlow & Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor; Peters et al. (1985) Monoklonale Antikörper: Herstellungund Charakterisierung [Monoclonal Antibodies: Preparation andCharacterization], Springer Verlag], fused with myeloma cells togenerate antibody-producing hybridoma cells, after which suitable clonesare isolated and cultured individually. Specific screening methods areused to select the clones which are producing the desired monoclonalantibodies. In this connection, enzyme immunoassay, radio immunoassayand/or Western blotting is/are used to examine the specificity withwhich the antibodies which are released into the cell culturesupernatant bind to, for example, the immunizing antigen or to apossible carrier of the immunizing antigen. Hybridomas which produceantibodies according to the invention are propagated clonally. Thehybridoma cell lines which are obtained in this way are then availablefor producing monoclonal antibodies over an extended period. Relativelylarge quantities of antibody can be obtained, for example, from cellculture supernatants, in particular from fermentors or roller culturesas well as from ascites.

Depending on the desired purpose, it may be advantageous to use onlyparts of the antibodies, such as Fab, F(ab′)₂, or Fab′ fragments. Thelatter can be produced, for example, using the enzymic cleavage methodsknown to the skilled person [see also Harlow & Lane (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor].

The antigen-binding sites in an antibody are located in what are termedthe variable domains, which are encoded by the V genes. It is alsopossible to use the known recombinant methods [e.g. Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, 2nd edition; McCafferty et al. (1990)Nature 348: 552-554] to determine the corresponding nucleic acidsequence of an antibody according to the invention and, as a result, thecorresponding amino acid sequence as well, provided the latter had notalready been elucidated by means of amino acid sequencing. The hybridomacells or the antibody-producing immune cells of immunized animals can beused as starting material for these analyses.

Knowing the nucleic acid sequence and/or amino acid sequence, it is thenpossible to use customary recombinant and molecular biological methods[see also Johnson & Chiswell (1993) Current Opinion in StructuralBiology 3: 564-571] to prepare humanized, chimeric, bispecific oroligospecific antibodies, as well as complementarity determiningregion-derived peptides (“minimal recognition units”), single-chainfragments and/or functional fusion products, e.g. recombinantly preparedantibody-enzyme constructs [see also Larrick & Fry (1991) HumanAntibodies and Hybridomas 2: 172-189; Kitano et al. (1986) Appl.Microbiol. Biotechnol. 24:282-286; Thompson et al. (1986) J. Immunol.Methods 94:7-12] which bind to the FSAP MR I-specific epitope, inparticular to a peptide according to the invention. These peptides whichare encompassed in the term “antibody” can be used, for example, toachieve a decrease in immunogenicity and/or an increase in activity inconnection with administration as a pharmaceutical or an in-vivodiagnostic agent, and/or advantages ensue for use of the peptides as orin an in-vitro diagnostic agent. The antibodies can also be prepared,where appropriate with the aid of recombinant methods, in fungi such asyeast cells [Fischer et al. (1999) Biol. Chem. 380: 825-839; Hiatt etal. (1992) Genetic Engineering 14: 49-64], in plant, animal andprokaryotic cells (see also WO 95/25172) as well as in isolated humancells.

This invention furthermore also relates to fungi, animal, plant orprokaryotic cells, as well as isolated human cells, which produce anantibody according to the invention. A preferred embodiment of thisinvention encompasses hybridoma cell lines which produce the antibodiesaccording to the invention, for example the hybridoma cell lines a)ECE-KLH 2004-9/014, b) ECE-KLH 2004-9/026, c) ECE-KLH 2004-35/05, d)ECE-KLH 2004-34/08, and e) ECE-KLH 2004-151/013, which have beendeposited in the DSMZ—Deutsche Sammlung von Mikroorganismen andZellkulturen [German collection of microorganisms and cell cultures]GmbH, Mascherode Weg 1b, 38124 Braunschweig, Germany, under the receiptnumbers a) DSM ACC2675, b) DSM ACC2676, c) DSM ACC2674, d) DSM ACC2725and e) DSM ACC2726.

The examples which are described below serve to illuminate individualaspects of this invention by way of example and are not to be understoodas being a limitation.

EXAMPLES Example 1 Preparing FSAP Marburg I-Specific MonoclonalAntibodies

1a) Immunizing Mice

BALB/c mice were in each case immunized intraperitoneally with 20 μg ofimmunizing antigen [KLH-bound peptide having the amino acid sequenceSer-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID No: 3)] inImmune Easy Mouse adjuvant (Qiagen GmbH, Germany). After 4 and 8 weeks,the mice were given a booster injection with in each case 20 μg ofimmunizing antigen without adjuvant. For the last 3 days prior to thefusion, the mice were boosted intravenously with in each case 10 μg ofimmunizing antigen.

1b) Fusion

After the mice had been killed by CO₂ inhalation, the spleens wereremoved and single-cell suspensions were prepared in serum-freeDulbecco's modified Eagle medium (DMEM; PAN Biotech GmbH, Germany). Thecells were centrifuged (652×g) and washed 2× in DMEM. The cell count wasthen determined by means of Trypan Blue staining. 2×10⁷ myeloma cells(Sp2/0) were added to about 10⁸ spleen cells. Following centrifugation(360×g), the supernatant was discarded and 1 ml of polyethylene glycolsolution (PEG 4000, Merck Eurolab GmbH, Germany; approx. 50% strength inDMEM) was added to the cell pellet which, after resuspension, wasincubated at 37° C. for 1 minute. Approx. 10 ml of DMEM were then addedand the mixture was incubated at room temperature for from 2 to 4minutes. The fused cells were centrifuged off (326×g) and the pellet wasresuspended in DMEM+10% fetal calf serum (Bio Whittaker Europe,Belgium)+HAT medium (CC Pro GmbH, Germany) and aliquoted into 24-wellcell culture plates (Corning Costar GmbH, Germany). The approximate cellconcentration was 5×10⁴ to 5×10⁶ cells per well.

After 2 to 3 weeks, the cell colonies (hybrids) which developed wereremoved and transferred to new culture plates.

1c) Screening

The specificity of the antibodies which were released into the cellculture was tested in a first test step using microtiter plates (NuncGmbH & Co. KG, Germany) which were coated with a peptide having theamino acid sequence Ser-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr(SEQ ID No: 3).

100 μl of cell culture supernatant (diluted 1:2) were pipetted into eachwell of the microtiter plate and the plate was incubated at from +15 to+25° C. for 1 hour. After the plate has been washed twice with PODwashing solution (OSEW; Dade Behring Marburg GmbH, Germany), 100 μl ofanti-mouse IgG/F(ab′)₂-POD conjugate (Dade Behring Marburg GmbH,Germany) were aliquoted into each well and the plate was incubated atfrom +15 to +25° C. for 1 hour. After the plate has been washed afurther two times, 100 μl of Chromogen TMB solution (Dade BehringMarburg GmbH, Germany) were aliquoted into each well and the plate wasincubated at from +15 to +25° C. for a further 30 minutes. After theincubation, 100 μl of POD stop solution (Dade Behring Marburg GmbH,Germany) were aliquoted into each well and the microtiter plate wasevaluated at 450 nm in a BEP II (Behring-ELISA Processor II, DadeBehring Marburg GmbH, Germany).

In a second test step, the hybrids were examined once again in the sametest format, as described above, after having been separated intoindividual cells.

1d) Cloning

Individual cells of hybrids which produce FSAP MR I-specific antibodieswere cloned using a micromanipulator (Leitz Messtechnik GmbH, Germany).Culture supernatants from these clones were purified as described under1g) and characterized in more detail as described under 1e), 1h) and1i). Antibodies according to the invention which bind to the FSAP MRI-specific epitope are produced, for example, by the clones

a) Mab (mouse) directed against ECE-KLH 2004-9/014(1)

b) Mab (mouse) directed against ECE-KLH 2004-9/026(2),

c) Mab (mouse) directed against ECE-KLH 2004-35/05(1),

d) Mab (mouse) directed against ECE-KLH 2004-34/08(2), and

e) Mab (mouse) directed against ECE-KLH 2004-151/013(2).

These hybridoma cell lines were deposited in the DSMZ-Deutsche Sammlungvon Mikroorganismen and Zellkulturen [German collection ofmicroorganisms and cell cultures] GmbH, Mascherode Weg 1b, 38124Braunschweig, Germany, under the receipt numbers a) DSM ACC2675, b) DSMACC2676, c) DSM ACC2674, d) DSM ACC2725 and e) DSM ACC2 726.

1e) Determining the Antibody Subclasses

The subclasses of the antibodies directed against the FSAP Marburg Ivariant were determined using the IsoStrip™ mouse monoclonal antibodyisotyping kit supplied by Boehringer Mannheim, Germany. The followingsubclasses were determined:

a) Mab (mouse) directed against ECE-KLH 2004-9/014 (1)-subclass: IgG 2a,

b) Mab (mouse) directed against ECE-KLH 2004-9/026 (2)-subclass: IgG 2a,

c) Mab (mouse) directed against ECE-KLH 2004-35/05 (1)-subclass: IgG 2b,

d) Mab (mouse) directed against ECE-KLH 2004-34/08 (2)-subclass: IgG 1,

e) Mab (mouse) directed against ECE-KLH 2004-151/013(2)-subclass: IgG 1.

1f) Producing the Antibodies

In order to produce relatively large quantities of antibody, thecorresponding cell clones are transferred to roller flasks (CorningCostar GmbH, Germany) and expanded at +37° C. to the desired finalvolume. After that, the roller culture suspension is filtered through0.22 μm in order to remove the cells. The antibody solution, which isnow cell-free, is concentrated using an ultrafilter (cut-off point 30000dalton) and then purified.

1g) Purifying the Antibodies

The resulting antibody solution is rebuffered with 0.14 M phosphatebuffer, pH 8.6, and loaded onto a chromatography column which is filledwith rProtein A Sepharose™ Fast Flow (Amersham Biosciences Europe GmbH,Germany) (1 ml of rProtein A Sepharose™ Fast Flow is used per 10 mg ofantibody to be purified). All the unbound components are removed bywashing the column with 0.14 M phosphate buffer, pH 8.6. The boundantibody is eluted from the column with 0.1 M citric acid, pH 3.0, anddialyzed against 0.05 M sodium acetate+0.5 M NaCl+0.05 M Tris+0.01%sodium azide, pH 7.0.

1h) Selecting Antibodies which are Suitable for an FSAP Marburg ISandwich ELISA

The reaction of the monoclonal anti-FSAP Marburg I antibodies with theFSAP MR I-specific epitope [peptide having the amino acid sequenceSer-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID NO: 3)] orthe corresponding FSAP wild-type epitope [peptide having the amino acidsequence Ser-Trp-Gly-Leu-Glu-Cys-Gly-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID NO:6)] was examined:

Reaction with an FSAP MR I-Specific Peptide:

A microtiter plate which is coated with a peptide having the amino acidsequence Ser-Trp-Gly-Leu-Glu-Cys-Glu-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID NO:3) is used as the solid phase. The anti-FSAP MR I antibodies fromculture supernatants are incubated on it. After a washing step, aconjugate consisting of rabbit polyclonal anti-mouse antibodies and theenzyme peroxidase is used, together with a subsequent color reaction, todetect binding of the antibody to the peptide.

Reaction with an FSAP Wild-Type Peptide:

A microtiter plate which is coated with a peptide having the amino acidsequence Ser-Trp-Gly-Leu-Glu-Cys-Gly-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID NO:6) is used as the solid phase. The anti-FSAP MR I antibodies fromculture supernatants are incubated on it. After a washing step, aconjugate consisting of rabbit polyclonal anti-mouse antibodies and theenzyme peroxidase is used, together with a subsequent color reaction, todetect binding of the antibody to the peptide.

Those antibodies which exhibited a reaction with the peptide having theamino acid sequence Ser-Trp-Gly-Leu-Glu-Cys-Glu-LyS-Arg-Pro-Gly-Val-Tyr(SEQ ID NO: 3) (FSAP Marburg I variant) but which, at the same time, didnot react with the peptide having the amino acid sequenceSer-Trp-Gly-Leu-Glu-Cys-Gly-Lys-Arg-Pro-Gly-Val-Tyr (SEQ ID NO: 6) (FSAPwildtype) were selected. The suitability of these antibodies for use assolid-phase antibodies in a sandwich ELISA using an FSAP MR I-specificconjugate antibody, which is coupled to horseradish peroxidase employinga method known to the skilled person (e.g. Nakane conjugation), wasinvestigated.

The suitability was examined in a sandwich ELISA as described in Example3a). The essential criterion for deciding on the suitability was anunambiguous differentiation between the FSAP Marburg I variant and theFSAP wild type, i.e. high signal generation with samples which containthe FSAP Marburg I variant and no, or only a low, signal generation withFSAP wild-type samples. Citrate plasma is the preferred sample type.Other criteria are the lower detection limit and the linearity of thecalibration curve.

The antibodies which were produced by the clones

a) Mab (mouse) directed against ECE-KLH 2004-9/014(1) (DSM ACC2675),

b) Mab (mouse) directed against ECE-KLH 2004-9/026 (2) (DSM ACC2676),

c) Mab (mouse) directed against ECE-KLH 2004-35/05 (1) (DSM ACC2674),

d) Mab (mouse) directed against ECE-KLH 2004-34/08 (2) (DSM ACC2725) and

e) Mab (mouse) directed against ECE-KLH 2004-151/013(2) (DSM ACC2726)

displayed the best results with regard to these criteria.

Example 2 Detecting the FSAP Marburg I Variant in a Sample

2a) Sandwich ELISA Test Method

Peroxidase-conjugated anti-FSAP antibodies were employed, in combinationwith the monoclonal anti-FSAP Marburg I antibodies according to theinvention, in an enzyme immunoassay performed in accordance with thesandwich principle.

During the first incubation, the FSAP Marburg I antigen variant which ispresent in the sample, provided it is indeed present, binds to theantibodies according to the invention which are directed against FSAPMarburg I and which are fixed to the surface of the wells of amicrotitration plate. After the wells have been washed out,peroxidase-conjugated anti-FSAP antibodies which are directed againstany arbitrary epitope of the FSAP MR I variant, or of the FSAP wild-typeprotein, such as those formed by one of the hybridoma cell lines DSMACC2453 and DSM ACC2454 (EP 1 182 258 A1), are used in a second bindingreaction. The excess enzyme-conjugated antibodies are washed out. Theenzyme activity which is bound in the wells is then determined. Theenzymatic reaction of hydrogen peroxide and tetramethyl-benzidine isterminated by adding dilute sulfuric acid. The color intensity, which isproportional to the FSAP MR I antigen concentration, is determinedphotometrically at a wavelength of 450 nm and either analyzedqualitatively using a cut-off or quantified using a calibration curveconstructed from standards. Such a sandwich immunoassay according to theinvention detects the FSAP Marburg I variant specifically in only onetest method.

The results are presented in Table 1. The significant color reaction inthe samples which are derived from heterozygous test subjects exhibitingFSAP Marburg I polymorphism demonstrates that using the antibodiesaccording to the invention makes it possible to determine the FSAPMarburg I variant specifically and reliably.

2b) LOCI® Test Method

The measurement principle of the LOCI® technology is based onchemiluminescence which is elicited by oxygen free radicals. Theprerequisite in this connection is that the reactive components (e.g.chemiluminescence latex particles and photosensitive latex particles)should be in spatial proximity, with this spatial proximity beingbrought about by the antigen-antibody complex.

A biotin-conjugated anti-FSAP antibody was used, in combination with themonoclonal anti-FSAP Marburg I antibodies according to the invention, ina homogeneous LOCI® performed in accordance with the sandwich principle.During the first incubation, the FSAP Marburg I antigen variant which ispresent in the plasma sample, if it is indeed present, binds to theantibodies according to the invention which are directed against FSAPMarburg I and which are fixed to the surface of chemiluminescenceparticles which contain an olefin. After the biotin-conjugated anti-FSAPantibody has been added, and after an incubation of 7 minutes at +37°C., photosensitive latex particles on whose surface streptavidin isfixed are added. If FSAP Marburg I antigen variant is present in thesample, anti-FSAP MR I-chemiluminescence particles, biotin-conjugatedanti-FSAP antibodies and photosensitive latex-streptavidin particlesthen form a spatially close complex. On excitation with light at awavelength of 680 nm, the photosensitive latex particles releaseshort-lived singlet oxygen, which now initiates aluminescence/fluorescence signal (520-620 nm) from the chemiluminescenceparticles which are bound by the antigen-antibody complex. As a resultof its instability, the singlet oxygen only reaches chemiluminescenceparticles which are located in the closest vicinity (<200 nm) to thephotosensitive latex particles. The emitted light is measured using amodified Tecan RSP 150 having an on-board luminescence detector unit[Ullman et al. (1994), Proc. Natl. Acad. Sci., 91: 5426-5430; Ullman etal. (1996) Clinical Chemistry, 42: 1518-1526].

2c) Detecting the FSAP Marburg I Variant in Patient Samples

The method described under 2a) was used to examine 10 plasma samples,while that described under 2b) was used to examine 84 plasma samples,from test subjects who had previously been genotyped. 5 samplesoriginated from test subjects who had a G/A transition at nucleotideposition 1601 of the coding region of the FSAP gene (where 1 is the A ofthe initiation codon) in one gene copy and were consequentlyheterozygous carriers of the FSAP MR I polymorphism which, at the aminoacid level, leads to a Gly/Glu amino acid substitution at position 534in the proenzyme (Gly/Glu 534). 5 further samples originated from testsubjects who had no mutation at nucleotide position 1601 of the codingregion of the FSAP gene but instead exhibited the wild-type genesequence (G) in both gene copies.

As can be seen from Tables 1 and 2, using an antibody according to theinvention in a sandwich ELISA or LOCI® assay makes it possible todifferentiate FSAP MR I-positive and FSAP MR I-negative samplesunambiguously. The monoclonal antibodies which are formed by thehybridoma cell lines DSM ACC2725 and DSM ACC2726 are particularlysuitable for being used in the LOCI® assay. A signal of 300 000 countswas specified as being the cut-off value in the LOCI® assay.

TABLE 1 Genotype at nucleotide Sample No. position 1601 OD₄₅₀ nm 10048G/G 0.011 10032 G/G 0.015 10033 G/G 0.016 10029 G/G 0.021 10045 G/G0.015 14942 G/A 1.625 14943 G/A 1.545 7020552 G/A 1.723 10047 G/A 1.5337020538 G/A 1.441

TABLE 2 Sample Genotype at nucleotide ID pos. 1601 Counts 1 G/G 8900 2G/A 1750000 3 G/G 12500 4 G/G 4800 5 G/G 9800 6 G/G 19300 7 G/G 22100 8G/G 35100 9 G/G 8900 10 G/G 7800 11 G/G 6600 12 G/G 8800 13 G/G 4100 14G/G 7700 15 G/G 8500 16 G/G 4700 17 G/G 8900 18 G/G 5600 19 G/G 7500 20G/G 6900 21 G/G 15300 22 G/G 14200 23 G/G 8600 24 G/G 5600 25 G/G 450026 G/G 7700 27 G/G 5900 28 G/G 7500 29 G/G 7300 30 G/G 9600 31 G/G 1340032 G/G 7600 33 G/G 8500 34 G/G 7700 35 G/G 6400 36 G/G 5600 37 G/G 760038 G/G 9100 39 G/G 7300 40 G/G 9800 41 G/G 4900 42 G/G 8500 43 G/G 2540044 G/G 12400 45 G/G 31200 46 G/G 22200 47 G/G 8600 48 G/A 1821000 49 G/G5300 50 G/G 7600 51 G/G 9400 52 G/G 5900 53 G/G 7800 54 G/G 8800 55 G/G24500 56 G/G 21500 57 G/G 27400 58 G/G 23300 59 G/G 14600 60 G/G 7300 61G/G 5600 62 G/G 9600 63 G/G 6700 64 G/G 21400 65 G/A 1250000 66 G/G 830067 G/G 7900 68 G/G 6900 69 G/G 4900 70 G/G 8800 71 G/G 15400 72 G/G18800 73 G/G 7700 74 G/G 8300 75 G/G 5700 76 G/A 1479000 77 G/G 7800 78G/G 8800 79 G/G 9400 80 G/G 5800 81 G/G 6400 82 G/G 7600 83 G/G 8200 84G/G 6900

1. An antibody produced by a hybridoma cell line chosen from DSMACC2674, DSM ACC2675, DSM ACC2676, DSM ACC2725, and DSM ACC2726.
 2. Theantibody of claim 1, wherein the antibody is linked to at least one of asolid phase and a component of a signal-generating system.
 3. A cellline which produces the antibody of claim 1, comprised of isolated cellschosen from animal, plant, fungal, and prokaryotic cells.
 4. The cellline of claim 3, wherein the isolated cells are hybridoma cells.
 5. Thecell line of claim 4, chosen from DSM ACC2674, DSM ACC2675, DSM ACC2676,DSM ACC2725, and DSM ACC2726.
 6. A method of separating moleculescomprising the amino acid sequence of SEQ ID NO:1 from other molecules,comprising: linking at least one antibody of claim 1 to a solid phase;contacting the solid phase with a sample that may comprise at least onemolecule comprising the amino acid sequence of SEQ ID NO:1; washing thesolid phase; and eluting the at least one molecule comprising the aminoacid sequence of SEQ ID NO:1 from the solid phase.
 7. A method ofdetecting at least one FSAP Marburg I variant in a sample, comprising:providing a sample which may comprise at least one FSAP Marburg Ivariant antigen; contacting the sample with at least one antibody ofclaim 1, wherein the antibody is linked to a signal generating systemcomprising a label; and detecting a signal from the specific binding ofthe antibody to the at least one FSAP Marburg I variant antigen with thelabel, thereby detecting an FSAP Marburg I variant antigen in thesample.
 8. The method of claim 7, wherein the at least one FSAP MarburgI variant comprises the amino acid sequence of SEQ ID NO:3.
 9. Themethod of claim 7, wherein the antibody is linked to a solid phase. 10.The method of claim 7, wherein the signal from the label is quantitated.